Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-021-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2024
Objective:
African Swine Fever (ASF) is a large DNA virus of approximately 180kb, and contains approximately 160 different open reading frames (ORF), with little or no function. The primary target cell in swine is macrophages. To identify a known function for these genes one methodology is to identify the host proteins that interact with the various ASFV ORFs. However, currently there is no yeast library that is derived from swine macrophage cDNA. To further complicate things many of the ASFV proteins have membrane localization or membrane insertion signals. Membrane proteins are very difficult to study protein-protein interactions. A system, using for identifying membrane protein interactions, is needed that is compatible for use for swine proteins. The goal of this project is to design a complete genome membrane yeast two hybrid system compatible to identify protein interactions in a swine system, and validate these interactions do occur in mammalian cells, and during viral infection.
Approach:
To develop a yeast library, particular attention will have to be on collecting RNA from primary cells, analyzing the RNA for suitable gene expression, and inserting the correct sequences into an appropriate vector for both yeast and bacterial expression. The library will be grown, while maintaining diverse and equal population of all proteins, and validated for use in African Swine Fever (ASF) research. Once preliminary studies are done, the results will be validated first in swine cells to show effectiveness of the development of the yeast library. As a majority of the ASF viral proteins have a predicted transmembrane domain and are likely membrane proteins, the next stage of this project will involve developing a membrane yeast-two hybrid (MYTH), based on the split ubiquitin system. This system has never been tested for swine proteins and its use for this application will be developed. Using the same methodology as for the swine macrophage yeast-two hybrid library, swine macrophage proteins will be inserted into the MYTH. Once inserted into the MYTH, they will be tested for diversity and tested for reactivity to ASFV proteins. Once this is accomplished the results will be validated in swine cells. In the next stage of the project the results will be validated during ASFV infection of swine macrophages, then we will evaluate the effectiveness of using a swine library in yeast to correlate to ASFV protein interactions that occur in swine macrophages.
